The present invention relates to the method for mass propagation of adventitious roots of ginseng, camphor ginseng, and wild ginseng by tissue culture and the improvement of their saponin content. More specifically the present invention relates to a method for improvement of saponin content and mass propagation of adventitious roots of ginseng, camphor ginseng, and wild ginseng by tissue culture which can produce in vitro adventitious roots that have excellent commercial value and effectiveness. This product was produced by including adventitious roots from callus, which was formed by tissue culturing of the leaf, root and stem of cultivated ginseng, camphor ginseng, and wild ginseng. After inducing the adventitious roots, it was mass propagated by shaking incubation or bioreactor incubation. In the mass propagation process, the optimal cultivating conditions which the saponin contents in the cultivated ginseng or wild ginseng yields almost the same contents in the natural ginseng were identified.
Botanically, ginsengs belong in Panax genus of Araliaceae family and their roots are used for medical purposes.
Globally, 6-7 plant species are known to belong to this species. Also, 3 types of the ginsengs are cultivated economically and traded in the ginseng market.
Geographically, ginseng whose plant name is xe2x80x9cPanax ginseng C.A. Meyerxe2x80x9d is distributed and cultivated at Far East Asia and was used for a tonic medicine, which is traditionally the most important oriental herb medicine in china.
Ginseng has been known to have excellent effects in curing and promoting recovery from diverse diseases and a broad research on ginseng""s drug action component and pharmacological effect have been conducted.
Until now, the scientifically proved important major effect is the maintenance of homeostasis of body control function. Based on this function, anti-fatigue and anti-stress effect, anti-diabetes effect, blood pressure control effect, anti-cancer effect, prevention of arteriosclerosis and high blood pressure, brain function fortification, gastrointestinal function reinforcement, immunity reinforcement, anti-virus effect are to be reported.
The primary effective components of ginseng are saponin, sapogenin, polyacetylene, pyragen derivatives, maltol.
However, it has some uneconomical aspects due to the facts that the natural ginseng, which has medical purposes, requires a long-term cultivation at the well drainage and cool highlands for 4-6 years. Also, it will be affected by the natural climate and will not be able to crop continuously, thus appearing uneconomically.
Accordingly, the plant tissue cultivating research has been studied to produce mass propagation of the precious medical ginseng all year around without any impaction of the climate. The method is to produce a ginseng tissue that has as much effective component as the natural medical ginseng by cultivating an indeterminate form of callus, which is obtained by culturing the root of the natural medical ginseng, in various nutrient media under different environmental conditions.
The technology of the mass propagating method of ginseng trichoid root (hairy root) has been registered as a domestic patent under the public patent number of 1993-0000004.
At present, the technology for tissue culturing of Korean ginseng, American ginseng and Tenchi ginseng for multipurpose is commonly practiced. In particular, the cultivation of callus, which was obtained from culturing various tissues of ginseng, organ differentiation and the successful cell culture of callus, are reported in the professional journals. However these experiments are mainly confined in the analysis of the impacts of the physical and chemical factors on the propagation of callus and cell. But not so many experiments have been reported on the cultivation of adventitious roots of ginseng. The utilization of ginseng requires a lot of effort including a long growing time of 5-6 years or at least 3-4 years and the difficulty of rotating crops. Nevertheless the demand of ginseng is ever growing for the usage in foods, cosmetics and medicine etc.
The present invention was invented based on the above-mentioned facts. The object of the present invention is to examine closely to the factors that affect the mass propagation of adventitious roots of ginseng, camphor ginseng, and wild ginseng so as to produce the ginseng all year around in the growth controller through the tissue culture of adventitious roots of ginseng, camphor ginseng, and wild ginseng in a shaking incubator or in a bioreactor. In the mean time the present invention is to provide an improved method for increasing saponin content in the adventitious roots of ginseng, camphor ginseng, and wild ginseng compared with the cultivated ginseng and wild ginseng and to induce the level of the saponin composition ratio as same as the natural ginseng by finding the optimal cultivation condition. Therefore, the present invention makes it possible to produce high saponin contented ginseng that has high commercial value and effectiveness.
The other object of the present invention is to apply the present method of mass propagation of the adventitious roots to the high added value products including camphor ginseng, and wild ginseng so as to make it possible to provide and produce raw materials of foods and substitute medicines.
To achieve the above objects of the present invention, the method for mass propagating the adventitious roots of ginseng, camphor ginseng, and wild ginseng comprises the steps of:
Culturing the tissues of root, stem, leaf from ginseng, camphor ginseng, cultivated ginseng and wild ginseng and desertion callus;
Propagating the developed adventitious root that was deserted from callus; and
Mass culturing the propagated adventitious root in the bioreactor.
The method to improve the saponin content of tissue cultured adventitious root for the mass propagation comprises the steps of:
Preprocessing of the one of adventitious root, obtained from tissue culturing of the ginseng, camphor ginseng, and wild ginseng using a growth regulator;
Seeding the preprocessed adventitious root in the media containing jasmonic acid or methyl jasmonic acid;
Culturing the adventitious root under the light in a balloon shaped or conical shaped bioreactor; and
Transferring the adventitious root to nitrogen excluded media within 5-10 days before harvest.
The method for the improvement of saponin content of the tissue cultured adventitious root for the mass propagation is to increase the diol-saponin and the triol-saponin of the adventitious root to the level of those in natural ginseng, thereby providing adventitious roots that have enough commercial value and usefulness in their effects.
Therefore, the present invention makes it possible to produce organic ginseng that are not affected by environments, soils and various contamination by agricultural chemicals all year around in the laboratory or in the factory. Subsequently, this invention makes it possible to satisfy the consumer""s desire for consumption by mass propagating the highly recognized wild ginseng in the standardized cultivator.
The primary object of the present invention was to examine closely the factors that affect the propagation of the adventitious root. The secondary object is to cultivate the adventitious root in a bioreactor using the most effective media and to examine the affect of the type of the bioreactor and the amount of the air injection to the bioreactor on the growth rate so as to solve the technical problems associated with the industrialization.
Therefore, the best mode for carrying out the invention consists following contents.
A 2-3 mm2 sized section was obtained from one of the sterilized ginseng, camphor ginseng, and wild ginseng and was seeded on a MS media containing 1.0-10.0 mg/L of 2,4-D (2,4-dichlorophenoxy acetic acid), pichioram, NAA naphthaleneacetic acid) to induce callus. The most desirable effect could be obtained when the concentration of added chemicals was 2.0 mg/L.
After propagating the induced callus in a MS media containing 0.1-5.0 mg/L of 2.4-D as a growth regulator, it was subcultured every 2-4weeks Then, transferred to a MS media containing 1.0-5.0 mg/L of IBA or NAA to produce a adventitious root. Instead of using MS media, SH(Schenk and Hildebrandt) media, B5(Gamborg)media, LP(Quorin and Iepoivre)media and White media were used to propagate callus. But the similar effect was obtained except for the difference depending on the culturing period. Among them MS media and xc2xe SH media had the most desirable results. Also, a desirable result could be obtained when 1.0-5.0 mg/L of NAA of IBA was used as a growth regulator that affects callus growth.
The above adventitious root was propagated in MS media (ratio of inorganic matter to solvent is xc2xd to xc2xe, pH 5.7-6.0, sugar concentration 3-5%) at the temperature of 18-24xc2x0 C. The randomly sectioned newly formed lateral root including cultured explants in a size of 1-2 cm was seeded in a buoyant balloon type bioreactor and was cultured in a MS media containing 3% sugar under the conditions of 0.05-0.3 vvm of air injection at 22xc2x0 C. and pH 6.0 with a growth regulator in an amount of 1.0-10.0 mg/L BSAA (benzo[b]selenienyl acetic acid) or IBA or NAA.
It is desirable to increase the air injection amount in every 2 weeks to prevent the root tangling and to re-seed the adventitious root after 2 weeks of incubation in a bioreactor to prevent rate decrease. It could be possible step by step to mass propagate by scaling up to a 20-50 ton of cultivating adventitious roots using a bigger bioreactor.